Method and kit for sample preparation and endotoxin determination

Abstract

The invention relates to a method for preparation of a sample ( 10 ) of a formulation ( 11 ) for subsequent endotoxin determination, the formulation ( 11 ) suspected of comprising an endotoxin, the formulation ( 11 ) preferentially being a pharmaceutical formulation. The method comprises the following steps: application of the sample ( 10 ) to an endotoxin-free centrifugation column ( 2 ) containing a size exclusion chromatography matrix ( 5 ) that has been equilibrated with a suitable equilibration buffer ( 6 ) and elution of a flow through ( 15 ) of the sample by centrifugation, which flow through ( 15 ) can then be used for endotoxin determination. The equilibration buffer ( 6 ) is selected according to a subsequently used method of endotoxin determination, the equilibration buffer ( 6 ) only containing components not interfering with subsequently used method of endotoxin determination. Furthermore, the invention relates to a kit ( 20 ) for preparation of a sample ( 10 ).

I claim: 
     
       1. A method for preparation of a sample ( 10 ) of a formulation ( 11 ) for subsequent endotoxin determination:
 applying a sample ( 10 ) onto an endotoxin-free centrifugation column ( 2 ) containing a size exclusion chromatography matrix ( 5 ) that has been equilibrated with an equilibration buffer ( 6 ), wherein the matrix ( 5 ) has size exclusion volume or exclusion cut-off within the range of 2,000 Dalton to 20,000 Dalton and a high mechanical stability at centrifugation forces of up to 1,800 g; and 
 eluting a flow through ( 15 ) of the sample by centrifugation, wherein centrifugation separates monomeric LPS, large LPS complex, or LPS aggregate from small components of the formulation, the small components remain with the matrix ( 5 ), and the monomeric LPS, large LPS complex, or LPS aggregate are in the flow through ( 15 ), 
 wherein the flow through ( 15 ) can then be used for endotoxin determination. 
 
     
     
       2. The method of  claim 1 , further comprising removing excess equilibration buffer ( 6 ) prior to applying the sample ( 10 ) onto the centrifugation column ( 2 ) by centrifugation. 
     
     
       3. The method of  claim 1 , wherein the matrix ( 5 ) is an uncharged, hydrophilic gel matrix or wherein the matrix ( 5 ) is a crosslinked polyacrylamide gel matrix. 
     
     
       4. The method of  claim 1 , wherein the equilibration buffer ( 6 ) comprises a buffer substance that differs from a buffer used in a formulation ( 11 ), and wherein the equilibration buffer ( 6 ) comprises at least one bivalent cation. 
     
     
       5. The method of  claim 4 , wherein the equilibration buffer ( 6 ) comprises Ca 2+  or Mg 2+  in a concentration between 1 mM and 100 mM or wherein the pH of the equilibration buffer ( 6 ) is 6.0 to 8.5. 
     
     
       6. The method of  claim 5 , wherein the equilibration buffer ( 6 ) comprises an amphiphilic substance in a concentration below its critical micelle concentration, or equal or below its solubility threshold in the equilibration buffer ( 6 ) system. 
     
     
       7. A method for endotoxin determination within a sample ( 10 ) of a formulation ( 11 ):
 applying a sample ( 10 ) onto an endotoxin-free centrifugation column ( 2 ) containing a size exclusion chromatography matrix ( 5 ) that has been equilibrated with an equilibration buffer ( 6 ), wherein the matrix ( 5 ) has a size exclusion volume or exclusion cut-off within the range of 2,000 Dalton to 20,000 Dalton and a high mechanical stability at centrifugation forces of up to 1,800 g; 
 eluting a flow through ( 15 ) by centrifugation, which flow through ( 15 ) is collected, wherein centrifugation separates monomeric LPS, large LPS complex, or LPS aggregate from small components of the formulation, the small components remain with the matrix ( 5 ), and the monomeric LPS, large LPS complex, or LPS aggregate are in the flow through ( 15 ), and 
 testing the collected flow through ( 15 ) for endotoxin. 
 
     
     
       8. The method of  claim 7 , further comprising incubating the collected flow through ( 15 ) at a temperature for a time period prior to the testing step. 
     
     
       9. A kit ( 20 ) for preparation of a sample ( 10 ) of a formulation ( 11 ) to be used for subsequent endotoxin determination comprising the following endotoxin-free components:
 at least one centrifugation column ( 2 ) prepacked with a size exclusion chromatography matrix ( 5 ) or at least one centrifugation column ( 2 ) and a size exclusion chromatography matrix material, wherein the matrix ( 5 ) has size exclusion volume or exclusion cut-off within the range of 2,000 Dalton to 20,000 Dalton and a high mechanical stability at centrifugation forces of up to 1,800 g; and 
 an equilibration buffer ( 8 ). 
 
     
     
       10. The kit ( 20 ) of  claim 9 , wherein the equilibration buffer ( 6 ) comprises a buffer substance that is different from the buffer used in a formulation ( 11 ), and wherein the equilibration buffer comprises at least one bivalent cation. 
     
     
       11. The kit ( 20 ) of  claim 10 , wherein the matrix ( 5 ):
 is an uncharged, hydrophilic gel matrix, 
 is a crosslinked polyacrylamide gel matrix, 
 is non-collapsible at centrifugation forces of up to 1,800 g, 
 has an average particle size between 5 μm and 250 μm, or 
 has a size exclusion cut-off of less than 20 kDa, or 
 wherein the equilibration buffer contains 20 mM Tris/HCl at pH 7.4, 50 mM NaCl and 1 mM to 5 mM Ca 2+  or Mg 2+ , or contains 20 mM HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) at pH 7.2, 50 mM NaCl and 20 mM to 50 mM Ca 2+  or Mg 2+ . 
 
     
     
       12. The method of  claim 2 , wherein the removing step is performed with an applied centrifugation force that is the same as is applied during the eluting step and wherein the applied centrifugation force is more than 200 g. 
     
     
       13. The method of  claim 5  wherein the equilibration buffer ( 6 ) comprises Ca 2+  and/or Mg 2+  in a concentration between 20 mM and 50 mM or wherein the pH of the equilibration buffer ( 6 ) is 7.0 to 8.0. 
     
     
       14. The method  claim 6  wherein the amphiphilic substance comprises Lauryl alcohol, Tween 20, Polypropylenglycol, or SDS. 
     
     
       15. The kit ( 20 ) of  claim 11  wherein the matrix ( 5 ) has an average particle size between 5 μm and 50 μm. 
     
     
       16. The kit ( 20 ) of  claim 11  wherein the matrix ( 5 ) has a size exclusion cut-off of less than 6,000 Dalton. 
     
     
       17. The method of  claim 1 , wherein the matrix has a size exclusion volume or exclusion cut-off within the range of 4,000 Dalton to 7,000 Dalton. 
     
     
       18. The method of  claim 17 , wherein the matrix has a size exclusion volume or exclusion cut-off below 6,000 Dalton. 
     
     
       19. The method of  claim 7 , wherein the matrix has a size exclusion volume or exclusion cut-off within the range of 4,000 Dalton to 7,000 Dalton. 
     
     
       20. The method of  claim 19 , wherein the matrix has a size exclusion volume or exclusion cut-off below 6,000 Dalton.